Targeted and untargeted metabolomics and lipidomics in dried blood microsampling: Recent applications and perspectives

Abstract Blood microsampling (BµS) offers an alternative to conventional methods that use plasma or serum for profiling human health, being minimally invasive and cost effective, especially beneficial for vulnerable populations. We present a non‐systematic review that offers a synopsis of the analytical methods, applications and perspectives related to dry blood microsampling in targeted and untargeted metabolomics and lipidomics research in the years 2022 and 2023. BµS shows potential in neonatal and paediatric studies, therapeutic drug monitoring, metabolite screening, biomarker research, sports supervision, clinical disorders studies and forensic toxicology. Notably, dried blood spots and volumetric absorptive microsampling options have been more extensively studied than other volumetric technologies. Therefore, we suggest that a further investigation and application of the volumetric technologies will contribute to the use of BµS as an alternative to conventional methods. Conversely, we support the idea that harmonisation of the analytical methods when using BµS would have a positive impact on its implementation.


What is a microsampling technique?
There is an increasing need for alternative specimens for profiling of human health.Traditionally for assessment of health and disease, plasma and serum are used.However, the rather large amount of sample required makes them less useful for repeated sampling in tight intervals or vulnerable groups, such as infants or the elderly.Blood microsampling (BµS) is associated with the collection of typically less than 100 µL of dried or liquid blood samples on absorbing materials.
The collection varies depending on the technique, requiring capillary blood droplets from a lancet prick or by capillary/tube absorption. 1 Dried blood spots (DBSs) have been used in newborn screening (NBS) since 1961 with Robert Guthrie and his test to detect phenylketonuria (PKU) at an early stage in newborn blood. 2 Nowadays, standard DBS cards are still called Guthrie cards and are commonly used for NBS.In 2022, the USA, Europe and Latin America were the regions with the highest percentage of newborns screened (100, 78 and 32%, respectively). 3mpared with venous blood, DBSs are patient-friendly samples.
Indeed, DBS samples only need a small amount of blood (typically < 100 µL) 1 and are considered minimally invasive.Additionally, DBS collection for most people is not experienced as painful, 4,5 particularly advantageous for children and people scared of needles.The absence of a need for a phlebotomist or a needle, along with the elimination of centrifugation to separate plasma from blood cells, contributes to the simplicity of the sample collection of those devices.
Patients can self-collect samples at home by pricking one of their fingers and depositing blood drops on the designated card.After a 4-h drying period, the DBS card can be sent via standard mail to the analysing laboratory.Additionally, the COVID-19 pandemic highlighted a growing acceptance of home testing and self-sampling, preventing many individuals from visiting hospitals.Due to the lockdown, the use of self-sampling methods, such as DBS cards, has increased these last years. 6Furthermore, DBS samples are easily stored and show good stability for many metabolites, which is helpful for blood banks. 7Since the samples are dried, the risk of biological contamination and infection is low. 1 To avoid moisture, which can lead to the degradation of analytes, DBS cards need to be stored with desiccant and avoid air.
As venous blood, DBSs can be used for several types of analyses as lipidomic, proteomic and metabolomic, with only one extraction protocol. 8DBS was the first BµS device reported to be analysed by metabolomic analysis, but the haematocrit (HCT) affects the size of the spot, the homogeneity of the sample (known as the 'haematocrit effect'), and hence the reliability of the quantitative analysis. 9The HCT is the volume percentage of red blood cells.The lower the HCT, the lower the viscosity: the blood will spread faster through paper fibres and will make the blood spot large, colourless and less homogenous.
With a high HCT, the blood spot will be smaller, more intense in colour and more homogenous. 10,11Punching disks of the same size for blood spots with varying HCT levels results in different volumes, leading to significant measurement and quantification errors. 11On a traditional DBS card, the spot is punched to remove it from the card.Multiple punches can be made in one DBS spot.But due to the haematocrit effect, depending on where the punch is made, the sample may be different.Other BµS technologies that collect fixed volumes have been developed to overcome the DBS limitations; still, studies using DBSs and new technologies evaluate their stability, reliability/accuracy and applicability. 12-14

Other BµS devices
Since DBSs have been shown to be a convenient sampling solution, it has been consistently developing over time.In parallel, different alternatives from BµS have been developed to overcome the aforementioned problems of DBSs.One of the main problems of DBSs, as already discussed above, is the 'haematocrit effect' . 15To have a more homogeneous sample from one DBS, it is possible to use pattern cards, where a drop of blood is applied in the middle of the cross-shaped pattern and the blood will spread uniformly across the card, creating four replicate punch zone. 16The HemaSpot™ HF device from Spot On Sciences 17 works with the same technology: two or three drops of blood are applied in the middle of the device.The blood will spread uniformly across the height blades, reducing the haematocrit effect between the height samples.
Another solution to overcome the haematocrit effect is the development of quantitative devices to collect an exact volume of capillary blood.The volumetric absorptive microsampling (VAMS) 18 can absorb a fixed volume (e.g., 10, 20 or 30 µL) of blood depending on the size of its tip via a capillary action.Other devices like qDBS Capitainer ® , 19 the hemaPEN ®20 or HemaXis™ DB10 21 have capillary channels to measure a fixed volume of blood, maintaining sample integrity for quantitative analysis.Two additional devices employed for quantitative sample collection, OneDraw™ 22 and Tasso-M20™, eliminate the need for finger-piercing, instead opting for application on the upper F I G U R E 1 Summary of advantages and disadvantages of blood microsampling (BµS) techniques.
arm.This approach not only enhances patient acceptability but also minimises discomfort compared with traditional venipuncture and finger-prick methods. 23Figure 1 offers an overview of the frequently used microsampling devices for dried blood collection, and Table 1 provides detailed information about the mechanisms involved in the sample collection process.Instead of creating new collection devices to overcome the haematocrit effect, it is also possible to develop tools to measure the HCT of the blood spot.It can be done optically, with a smartphone app, 11 reflectance spectroscopy, 24 near-infrared or ultraviolet-visible spectroscopy [25][26][27] or simply with a scanner and image analysis. 28

Applications of BµS in the targeted and untargeted analysis of small molecules
Many clinical tests rely on the measurement of small molecules (e.g., metabolites, drugs, etc., generally molecules with a mass < 1500 Da), often with single or only a handful of molecules detected simultaneously in one method.Analysis of metabolites is nowadays referred to as metabolomics and can be performed in a targeted manner, focusing on a specific subset of metabolites (e.g., one compound class or metabolic pathway), or untargeted, aiming for comprehensive analysis of as many metabolites as possible.The comprehensive analysis of lipids is called lipidomics and is often regarded as a sub-discipline of metabolomics.
Conceptually, metabolomics can be regarded as an extension of clinical chemistry, analysing more molecules.
Since its initial use in NBS, DBS sampling has evolved to analyse a wide range of endogenous and exogenous compounds.Additionally, untargeted analysis has also been performed.Recent applications of DBS samples include the analysis of small metabolites, such as vitamin D and opioids, as well as larger molecules like peptides and proteins. 29VAMS has also seen extensive implementation, particularly in studies related to COVID-19 analytes, immunosuppressants and anticonvulsants. 30For instance, VAMS has been used to study various endogenous metabolites, including creatinine, steroids, vitamins, phosphatidyl ethanols and amino acids, as recently reviewed. 18ditionally, advanced emerging technologies such as Capitainer ® B-Vanadate, hemaPEN ® and TASSO-M20™ have shown potential for applications such as caffeine, exercise and steroid monitoring. 31pidomic analyses follow the same pattern of evolution and development, from the early use of DBSs, for instance, in fatty acid oxidation studies, 32 to the implementation of quantitative and volumetric analysis with Mitra ® , 33 and recently the evaluation of new technologies like hemaPEN ®34 and TASSO-M20™, 35 in physical exercise studies with athletes and runners.Examples of the classes identified during different studies are fatty acids, polyunsaturated fatty acids, acylcarnitines, sphingolipids, glycerophospholipids, cholesterol, triacylglycerols, prenols, cholesterol esters and sterols. 36 2022, BµS was commonly employed in various biomedical applications, including animal studies, neonatal and paediatric studies, therapeutic drug monitoring (TDM), omics-based biomarker research and forensic toxicology.Key applications included the development and validation of novel methods, comparison of microsampling with conventional sampling and evaluation of different microsampling devices for optimal application. 1 However, ongoing research aims to address knowledge gaps in the implementation of microsampling, focusing on preanalytical and analytical conditions, stability's impact on sample metabolites and the potential for self-sampling and automation. 30,31is review focuses on dry BµS studies from 2022 and 2023, specif-TA B L E 1 Evolution of BµS devices and their characteristics, based on different advanced technologies.< 5% (2022)/Quantitative device.The device is placed onto the skin of the upper arm.When the button is pressed, a needle punctures the skin and the blood is drawn onto the sample pods using vacuum.
ically liquid chromatography-mass spectrometry (LC-MS) analysis of both targeted and untargeted metabolomics and lipidomics, showcasing the potential benefits of microsampling devices.

Endogenous molecules: endogenous disease biomarkers, NBS
Between 2022 and 2023, eight papers have been published contributing valuable insights into disease applications concerning targeted endogenous metabolite assays in blood samples using various microsampling devices.The focus of these metabolites primarily revolves around amino acids, lipids and small molecules associated with disease markers or NBS.
[39][40][41][42] Notably, Gao et al. 43 deviated from this trend by validating a method using VAMS.In the case of Carling et al., 44  .After a qualitative comparison, it was demonstrated that the use of volumetric blood collection devices (VBCDs) in the analysis of phenylalanine (Phe) and tyrosine (Tyr) in blood samples of PKU patients avoids pre-analytical challenges associated with DBS cards.These challenges include issues related to the volume of blood applied, the HCT effect and the quality of the sample, which had an effect on monitoring the disease. 44ncerning sample collection in DBS assays, commonly, a subpunch of 3.2 mm in diameter was performed to collect the blood applied, 37,39,40,42 except for Zhan et al., 38 who obtained two 5 mm sub-punches.In Carling et al. 44 tests, the sub-punch in conventional PerkinElmer-226 filter paper was 8 mm, while in volumetric DBSs (HemaXis™ DB10), it was 7 mm, and in Capitainer ® , it was 6 mm.The blood volume collected was approximately 20 µL in DBSs from each spot, as it cannot ensure the quantity of volume in each sub-punch.
Gao et al. 43 collected 20 µL in VAMS devices as well.However, Carling et al. 44 performed a volume comparison in volumetric and quantitative devices (HemaXis™ DB10, Neoteryx Mitra ® and qDBS Capitainer ® ): 20 and 50 µL.The authors demonstrated the lack of a significant volume effect on Phe or Tyr concentrations when using volumetric devices (p > 0.01). 44ring sample extraction, samples were placed in a 96-well plate or Eppendorf with the extraction solvent mainly using pure methanol, 37 methanol/water (80:20, v/v) 40,44 or acetonitrile (ACN)/methanol (1:1, v/v) 43,44 (others not specifying).This solvent included the labelled internal standard (IS).The following steps involved mixing techniques (e.g., shaking, vortex or sonication at room temperature) and centrifuge for 20 min to 1 h, except for Mak et al., 40 Wu et al. 37 and Gao et al. 43 which include a dry and reconstitution step with N 2 gas flow.
The main applications of targeted analysis using microsampling devices are disease biomarkers for diagnosis or monitoring and NBS.
Wu et al. 37 employed a combination of DBS sampling and direct injection mass spectrometry (MS) technology to analyse the blood metabolic profiles of 166 individuals, including gastric cancer patients and healthy controls.Given the limitations of existing low-sensitivity diagnostic and screening methods for gastric cancer, the identification and selection of highly specific and sensitive biomarkers for early detection and disease monitoring are of critical importance.They identified 23 parameters that were significantly altered in gastric cancer patients compared with healthy individuals.These metabolites were involved in various metabolic pathways, including nucleoside synthesis, glycolysis and lipid and amino acid metabolism, which play crucial roles in cell tumour proliferation and immunosurveillance.A panel of nine metabolites, comprising amino acids (Ala, Arg, Gly, Orn), carnitines (CA-OH, CAR 10:2) and related ratios (Tyr/Cit, Val/Phe, C5/C3) emerged as an effective tool for discriminating patients with gastric cancer from healthy individuals (AUC: 0.9318) with sensitivity and specificity.
These findings suggest that the selected metabolites hold promise as novel biomarkers for gastric cancer detection.In a subsequent step, the authors aim to investigate whether the levels of these metabolites correlate with different stages and severity of gastric cancer. 37 the other hand, NBS aims to identify infants with congenital metabolic disorders early in life, enabling timely intervention and improved outcomes.However, current NBS methods have limitations, including false positives and false negatives biomarkers referral rates.
Target metabolomics has emerged as a promising tool to overcome these issues associated to NBS. 40 Zhan et al. 38  showed that the method is robust, reliable and sensitive by evaluating the intra-day and inter-day precision, linearity, limit of quantification and recovery.These results indicate that the method is suitable for routine NBS for CAH. 38arling et al. 44 performed for the first time an evaluation of the accuracy, precision and effect of blood volume and HCT on the measurement of Phe and Tyr to improve monitoring of patients with PKU.
They used three different VBCDs (qDBS Capitainer®, Hemaxis-D™ B10 and Neoteryx Mitra® device) in comparison with conventional DBS cards, with mean biases between the two methods ranging from −0.3 to 1.2% for Phe and from −0.5 to 1.6% for Tyr.The authors not only found that the three VBCDs were more accurate and precise than DBS sampling for the measurement of Phe and Tyr, but they were also less affected by blood volume and HCT.These findings suggest that VBCDs have the potential to improve the monitoring of Phe and Tyr levels in patients with PKU. 44U was also assessed in the study conducted by Gao et al. 43 They performed an approach to validate a VAMS-dried blood method for measuring Phe levels in PKU patients and compared its results to those obtained from traditional venous blood samples.The study included 24 PKU patients who participated in a Phase 2 clinical trial.Phe levels were then measured in both method samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
The study evaluated the accuracy, precision, linearity, stability and matrix effects of the VAMS method compared with the conventional venous blood method.A difference was observed in both the Bland-Altman analysis (−4.25%) and the Passing-Bablok analysis (−4.49%).
However, this difference is considered clinically insignificant as it is within the acceptable range of 10-15% for monitoring blood Phe levels in PKU patients.The findings of this study suggest that the VAMS-dried blood method is a reliable and convenient alternative to traditional venous blood collection for measuring Phe levels in PKU patients.This method may have the potential to improve patient compliance with blood monitoring and lead to better outcomes for PKU patients. 43other study in newborns investigated the impact of preanalytical variables, such as gestational age, weight, gender and time of sampling, on metabolite concentration in DBS samples. 39The study included 610 reference DBS samples from healthy newborns to determine reference concentrations of amino acids, acylcarnitines and succinylacetone.
There was no significant effect of these variables on the concentration of any metabolites.These findings suggest that the reference intervals for these metabolites can be reliably used for interpreting DBS metabolomics data in neonates.They serve as robust references for metabolic disease screening using LC-QqQ-MS detector, regardless of gender, weight or gestational age. 39recent study provides valuable insights into the influence of maternal lifestyle factors, drug intake and supplements during pregnancy on the metabolic profile of newborns. 42The study included 109 mother-infant pairs and utilised NBS test to assess the impact of various factors such as smoking, alcohol consumption and physical activity.Maternal habits were evaluated through a questionnaire, and NBS results were collected from the Abruzzo regional NBS laboratory.
Flow injection analysis tandem mass spectrometry (FIA-MS/MS) was employed to analyse amino acids and acylcarnitines (CAR) in DBSs.The finding revealed that maternal smoking during pregnancy was asso-ciated with significantly higher concentrations of CAR 14:2 and CAR 18:2 in NBS samples.Conversely, increased maternal physical activity was linked to higher concentrations of glutamine and glutamate in newborns.However, maternal intake of iodised salt in diet was significantly related to a decreased level of free carnitine.While further research with larger and more diverse metabolic assessment methods is necessary, the study underscores the impact of maternal lifestyle on the metabolic health of newborns and emphasises the importance of being mindful of potential false positives during NBS. 42 their study, Mak et al. 40 validate a targeted metabolomics panel for second-tier NBS, evaluating its performance in identifying infants with four specific metabolic disorders: glutaric aciduria type 1 (GA1), multiple acylcarnitine deficiencies (MACD), methylmalonic acidemia (MMA) and propionic acidemia.The research included 156 DBS samples from infants with confirmed diagnoses of GA1, MACD, MMA or propionic acidemia, along with 194 DBS samples from healthy control infants. 40ing a rapid and high-throughput LC-MS/MS method with a 3 min runtime per sample and a triple-quadrupole mass spectrometer, the targeted metabolomics panel quantified 20 metabolites, including 16 acylcarnitines, three amino acids and succinylacetone.The results demonstrated high sensitivity and specificity for each of the four disorders.For GA1, the sensitivity was 100%, and the specificity was 99.5%.
For MACD, the sensitivity was 94.7%, and the specificity was 99.5%.
For propionic acidemia, the sensitivity was 100%, and the specificity was 99.5%.These high sensitivity and specificity values underscore the accuracy of the panel in identifying affected infants, thereby minimising false positives.This precision is crucial for preventing unnecessary family stress, medical interventions and healthcare extra costs. 40st, Kilgore et al. 41 aimed to develop a universal second-tier NBS LC-MS/MS method for amino acids, lysophosphatidylcholines and organic acids in DBS samples, addressing the limitations of current NBS methods.The method was validated using a panel of reference DBS samples and clinical DBS samples from infants with confirmed metabolic disorders.The developed LC-MS/MS method demonstrates high sensitivity, specificity and linearity for the quantification of those metabolites and was shown to be robust and reproducible with excellent intra-and inter-day precision. 41A summary of the sample collection, sample preparation and analytical method used in the publications included can be found in Tables 2 and 3.
A summary of the sample collection, sample preparation and analytical method used in the publications included can be found in Tables 4   and 5.

References
Targeted metabolomics

Therapeutic drug monitoring
After using BµS to look for disease biomarkers, researchers naturally stayed in the health field and started to look for the therapeutic drugs, their behaviour and the response of the metabolome.TDM helps to know if the medication dosage is adapted to the patient in order to hold a constant level of the drug, for example, an immunosuppressant after transplantation.
BµS is not yet used in routine TDM workflow, but several studies show an increasing interest in this type of microsampling devices. 45deed, microsampling devices need a small amount of blood, most analytes are stable even at ambient temperature, and since the sample is dry it is less biohazardous than wet blood. 1 Furthermore, since TDM requires repeated sampling in short intervals, BµS are of great interest.In the last 2 years, BµS has been studied in the monitoring of immunosuppressant drugs used after organ transplantation like tacrolimus and the associate biomarker creatinine, [45][46][47][48][49][50][51][52][53][54] anti-cancer such as PARP and kinase inhibitors, [55][56][57][58] antibiotics in children and neonates, [59][60][61] anti-tuberculosis drugs 62 and anti-fungal medication. 63

Antidoping
Urine is the most used matrix for routine drug testing.Indeed, most doping substances' metabolites are eliminated via the kidneys, 64 and it is a quick and non-invasive sample collection.Since 1992, blood has also been used in anti-doping routines in complement of urine analysis. 65World Anti-Doping Agency (WADA) and its accredited laboratories have been doing research on DBSs since 2000, 66 and WADA is currently thinking about implementing DBSs into the Athlete Biological Passport. 67Indeed, BµS allowed to improve monitoring of blood hormone and drug levels and help to support the evaluation of circulating drug concentrations during a competition (doi.org/10.1080/10408363.2022.2103085).
Anabolic androgenic steroids (AASs) are used by athletes to increase their muscle mass, physical strength and sportive performance as well as to recover from injury. 68,69Steroid esters are often used in doping since they can prolong the half-life of exogenously ingested endogenous AASs. 70Sadly, the abuse of steroid esters is not detectable directly in urine.The current method uses gas chromatography combustion isotope ratio MS -which is time and sample-consuming -and compares the results to the 'Steroidal Athlete Biological Passport' since the isotope ratio of natural and synthetic hormones are different. 71eroid esters are not stable in whole blood because they can be hydrolysed.Thankfully, the hydrolases are inactivated once the blood dries, 71 making DBSs the ideal matrix to analyse steroid esters directly. 72However, the volume of blood is small (<50 µL), and the polarity of steroid esters is low.Therefore, a derivatisation step is needed to increase the sensitivity. 68,71ring the XXXII Olympic Games and the XVI Paralympic Games in Tokyo, Japan, in 2021, DBSs were tested for steroid esters analysis.A total of 11 testosterone esters, nine nandrolone esters and six boldenone esters were investigated in 70 samples from different types of athletes (including aquatics, athletics, boxing, canoe, rowing, weightlifting and wrestling athletes).Since the WADA technical document for DBS analysis had not been approved before the game, 20 µL of whole blood was added to the DBS card in the laboratory instead of a real DBS sample collection by finger pricking.After drying, a punch of 6 mm was mixed with an IS solution, MTBE, isopropanol and methanol.The extracted solution was dried, and the residue was dissolved in methoxylamine and methanol.The sample was incubated before analysis with the LC-QqQ-MS. 72ring the XXIV Olympic Winter Games and the XIII Paralympic Winter Games in Beijing, China, in 2022, DBS samples were collected from the fingertips of the athletes for the first time.A collection and security kit called ShellCore was developed for the occasion. 73Six testosterone esters were analysed using LC-HRMS. 74The feedback from athletes about DBS sample collection was positive, so WADA TA B L E 5 Analytical methodology and instrumental analysis (2022-2023) -targeted exogenous metabolomics.would like to increase this kind of sample collection in the future without replacing the traditional ones. 75e WADA-accredited anti-doping laboratory of Rome transposed their anti-doping screening methods in urine 76 to DBSs.A total of 235 compounds (225 drugs and 10 main metabolites) were detected in a 20 min UHPLC run.Also, seven microsampling devices were tested, including DBS cards, VAMS, HemaSpot™ HF and Tasso-M20™. 77

Forensic
Blood is one of the most found biological traces on crime scenes.Lots of analysis can be done with it, including toxicology.In forensic studies, it can be interesting to develop analytical methods on DBSs.Indeed, in some cases, you cannot find people but their blood on the crime scene.
It is usually collected a long time after the crime, and it can be dry.With those bloodstains, it is possible to know if the person was under the influence of drugs, alcohol, medication or other substances.Since the exact volume of bloodstains is unknown, the volume can be estimated thanks to the IS versus endogenous amino acids ratio. 78Since the concentration of the drugs will only be estimated, the results must be taken cautiously.Plus, if the subject has abnormal amino acid levels, which can occur in certain diseases, the drug concentration will be underestimated or overestimated.But it can still give an idea of the state of the person at the moment of the bleeding.
In the case of car accidents, the driver is often injured.Their blood can be found on their cloth or in the car, and the bloodstains analyses can tell the driver's psychomotor performance at the time of the accident. 78Licit and Illicit drugs as morphine, methamphetamine and nordazepam are stable in dry bloodstains for at least 6 months. 79Benzodiazepines are not stable in whole blood and are easily hydrolysed. 80 dry blood, the enzyme that catalyses the hydrolysis is deactivated. 81prazolam and α-hydroxyalprazolam have been analysed after being stored for 17 years at room temperature. 82S cards can be used in forensics to stock samples to perform future analysis.Indeed, DBS cards need a small amount of blood, take less space than whole blood for storage (no need for a freezer) and can be stored for several years.Several analytes (like the one with an ester or amide group) are degraded in whole but not in dry blood, due to the inactivation of the enzyme that causes the hydrolysis. 80,81,83Plus, liquid blood is subjected to freeze thaw cycles that can degrade the analytes. 82study showed that stored at room temperature with a desiccant, 33 drugs including sedative hypnotics, antiepileptic drugs and antipsychotic drugs could still be detected with LC-MS/MS after 3-5 years.
Between 2016 and 2018, blood was collected from 105 drug poisoning cases and prepared as DBS cards.They were stored between 3 and 5 years at room temperature, protected from light and humidity in a sealed bag with a desiccant.The whole blood was analysed at the time of sample collection. 84ssano et al. 85 developed a method that can detect 132 drugs (synthetic opioids, cathinones, hallucinogens and fentanyl) in less than 10 min.They were able to detect and quantify those substances with concentrations between 5 and 100 ng/mL. 85

Per-and polyfluoroalkyl exposition
Per-and polyfluoroalkyl substances (PFAS) have been largely used since the 1940s in several fields (chemical, electronic, photographic, military, aviation, agricultural, packaging and textile applications).
Unfortunately, PFAS are hardly degradable, and therefore they have a high bioaccumulation level.Since the early 2000s in the USA, the concentration of PFAS in human blood has been tracked regularly because of their risk to human health. 86,87Currently, the PFAS screening is done through venous blood.Lin et al. 87 developed a method to analyse 22 PFAS in DBSs.Parallel to this method development, Koelmel et al. 88 use untargeted screening to detect novel perfluoroalkyl substances and analogues.A total of 28 PFAS and 86 potential PFAS were detected with their fragmentation pattern.
It is known that during pregnancy, a mother shares nutrients with the foetus, but also chemical contaminants. 89In 2023, Rosen Vollmar et al. 90 compared the level of thyroid hormone in newborns with their level of PFAS.Both thyroid hormone and PFAS analysis were made through DBSs, using the method developed by Lin et al. 87 The same year, Taibl et al. 91 studied the effect of prenatal PFAS exposition on the newborn metabolome via untargeted metabolomics.The PFAS analysis was conducted on the mother serum and the newborn metabolome on DBSs.

UNTARGETED METABOLOMICS
Untargeted metabolomics analyses employing BµS devices were used in several studies between 2022 and 2023.The studies included evaluations on sample storage as well as general development of the methodology and the identification of potential biomarkers associated with disorders in newborns and adults (for example, galactosemia,

very long-chain acylcarnitine dehydrogenase deficiency [VLCADD] and medium-chain acyl-coenzyme A dehydrogenase deficiency [MCADD]).
Most of the studies used DBSs, while only one study used VAMS. 12e DBS sample preparation and extraction used in most cases punching with a punch size of 3 mm -except for one study where 15 mm punches were used 91 -and extraction was performed with different mixtures of water with methanol and/or ACN by vortexing, agitating/shaking and/or sonicating on a total time from 30 to 120 min.
Three of the studies reported the use of an Orbitrap LC-MS/MS system, 40,91,92 and furthermore, one study implemented direct injection high-resolution MS. 93 Tables 6 and 7 show a summary of the set-ups used in the found studies.
The studies had different aims and settings, for example, optimisation of sample preparation or analysis of the stability of the specimens.Guo et al. 13 compared the effect of applying different volumes of blood (35 or 75 µL) on the absorbing material by taking central and outer 3 mm punches and the coverage using 1, 2 or 3 blood punches.They found higher concentrations of some metabolites (e.g., 12 metabolites including carnitine, adenosine monophosphate, palmitoleic acid and other lipids) when sampling in the centre than outside when using 75 µL of blood, while a more homogenous distribution when using 35 µL.
They interpreted that this was due to the chromatographic effects of the filter paper and a higher residence time with higher sample volumes.Furthermore, a single 3 mm punch reached more than 90% of the metabolite coverage of the three punches. 13reover, few studies tested the stability of blood micro samples. 7,12,13Guo et al. 13 tested the stability for 1 year at 4 and −20 • C, sampling at 4, 14 and 365 days.The samples presented acceptable stability at 4 • C within just 4 days and at −20 • C within 14 days but not anymore for 365 days. 13Ottosson et al. 7 report on a long-term stability study with neonatal DBS samples stored at −20 • C for 10 years.They concluded that more than 71% of the DBS metabolome was stable, 7 most of the unstable metabolites were affected either by the biobank or the transportation storage, and only a few metabolites were strongly influenced by storage.Volani et al. 12 evaluated the short-term stability of VAMS samples from 2 h to 2 weeks at room temperature and 4 • C.They found that in the samples below 6 h, the metabolome varied less than 3%, from 1 day to 1 week 10% up to 60% and after 1 week of storage, the variations decreased. 12Overall, these results support the idea of storing DBSs at −80 • C for long-term storage. 13However, if there is no need to detect lipid metabolites, then long-term storage at −20 • C is a suitable option. 12Furthermore, samples should 'rest' in storage at −80 • C to decrease variation if they are not collected at the same time, for example, in a longitudinal study. 12Complementary stability studies are needed to understand the temperature and storage effects on different metabolite groups in other types of blood micro samples.
Guo et al. 13 also presented a validation of the analytical method evaluating reproducibility, linearity, the limit of detection and recovery.Complementary, they conducted a clinical validation on a metabolomics study of congenital hypothyroidism.The method had satisfactory stability, precision, linearity and recovery, with a limit of detection minimum from 0035 ng/mL for CAR 8:0-d3 to 26 ng/mL for salicylic acid, a linear range between 3 and 5 orders of magnitude, relative standard deviations below 15% at different concentrations, and less than 30% in intra-and inter-day replicates. 13When applying the method in the clinical trial, it allowed them to detect 249 metabolites qualitatively and quantitatively and to select 14 metabolites to differentiate between the control group and the patients with congenital hypothyroidism. 13lani et al. 12 additionally evaluated the qualitative and quantitative metabolic differences between samples from capillary and venous blood (whole blood and plasma) using VAMS.They found the largest overlap between capillary and whole blood, while the opposite for capillary blood and plasma.Quantitatively, metabolites detected in the three matrices had considerably different abundances between the matrices.Interestingly, the results for capillary samples presented the lowest reproducibility or larger variations.However, the authors still concluded that using VAMS with capillary blood samples can capture biological differences of interest not found in plasma samples. 12nversely, Schneider et al. 94 compared the use of DBSs with other body fluids and dried spots usually found in crime scenes aiming to find a universal biomarker and determine time since deposition.
They peak-picked 24,904 possible molecular features shared across all four body fluids.However, they concluded that each biofluid should be evaluated using independent biomarkers.From the blood-specific compounds, they verified that four of them (L-phenylalanyl-L-alanine, 3-indole acrylic acid, spermidine and argininosuccinic acid) could be used to monitor time since deposition dynamics over 4 weeks. 94o studies used untargeted metabolomics on DBSs to discover potential biomarkers for metabolic diseases associated with fatty acid β-oxidation: very VLCADD and MCADD. 95,96The authors compared DBS samples from newborns positively diagnosed with the specific disorder against healthy controls.The authors found distinctive metabolomics profiles, altered metabolic pathways and interesting potential metabolic biomarkers (3,4-dihydroxytetradecanoylcarnitine, PIP (20:1/PGF1alpha), and PIP2 (16:0/22:3) for VLCADD newborns, and glutathione and oxidised lipids such as PGP (a-21:0/PGF1alpha) in MCADD newborns).The results suggested that oxidative stress results from both diseases, increasing the concentration of glutathione as an antioxidant defense. 95,96The same group also investigated patients with galactosemia, a genetic disorder related to disturbed galactose metabolism, and found alterations in the metabolome (e.g., PA (8:0/LTE4) and ganglioside GT1c (d18:0/20:0)) measured from DBSs.Abbreviations: DI-HRMS, direct infusion high-resolution mass spectrometry; ESI, electrospray ionisation; FIA, flow injection analysis; HESI, heated electrospray ionisation; N/A, not apply; N/S, not specified; PNS, positive/negative ionisation switching; qToF, quadrupole time of flying mass spectrometer; timsToF, trapped ion mobility spectrometry-time of flying mass spectrometer.
However, the study populations in all of these studies were small (n = 14-15) and therefore these results need to be validated in larger cohorts.
Taibl et al. 91 used newborns' DBS untargeted metabolomics to explore the molecular mechanisms between prenatal exposure to four PFASs (perfluorohexane sulfonic acid, perfluorooctane sulfonic acid, perfluorooctanoic acid and perfluorononanoic acid) exposure and gestational age-at-birth outcomes.Metabolites linked to foetal PFAS exposure suggested alterations in redox homeostasis, tissue neogenesis and endocrine disruption.They could be responsible for the linked health consequences including reduced length of gestation. 91rthermore, Tobin et al. 92 also identified metabolic signatures associated with preterm birth from DBS collected from pregnant women with HIV during pregnancy and infants at birth.They analysed samples collected before antiretroviral initiation and during treatment with either zidovudine or a protease-inhibitor-based regimen.The authors suggest untargeted metabolomics as a tool for hypothesis generation and biomarker discovery in complex disease processes, based on potential compounds (urate, methionine sulfone, cortisone and 17α-hydroxypregnanolone glucuronide) and mechanisms found as potentially linked with preterm births. 92rteling et al. 93 used untargeted metabolomics to analyse DBS samples of children and adolescents with a syndrome associated with increased risk of neurodevelopmental phenotypes including autism spectrum disorders (ASD) and intellectual impairment.The untargeted analysis identified metabolic signatures (the authors highlighted proline as a prominent distinguishing biomarker) and the metabolites were associated with the phenotypic expressions of ASD and intelligence quotient impairment. 93erall, in these clinical investigations of metabolome using DBSs, untargeted metabolomics enabled the identification of novel potential biomarkers to differentiate patients from healthy controls.These results also increase the understanding of the diseases and the involved biological pathways.However, most of the studies were cross-sectional and relatively small.There is a need for more longitudinal studies with larger cohorts.

LIPIDOMICS
The association between BµS strategies and lipidomic analysis is still in its early stages of development, and there are not many research studies on their use and applications in this field, but they have become a promising method for less invasive blood collection means that they are gradually being considered as a promising and favourable method for lipidomic studies. 31,36,98ong the most relevant articles published, between 2022 and 2023, we have selected seven papers where six use LC-MS methods and 1 uses FIA-MS method.In this section, the information has been organised in Tables 8 and 9, which summarise the research studies from a methodological point of view, focusing on the device used, sample collection, preparation, instrumental analysis and also the main goals and applications of the selected lipidomic studies.Some of the BµS techniques already summarised in Table 1 have also been gradually contributing to the field of lipidomics, as noted in the previous reviews. 31,36,98e selected articles of the last year are in line with previous lipidomic studies, [99][100][101][102] where the collection of DBSs on filter paper remains the most used approach today in lipidomic studies. 41,103,104en analysing these DBS card samples, it is assumed that the blood and analytes spread homogeneously.However, it is important to mention that DBS has a matrix effect dependent on the type of lipid and can cause interference in extraction efficiency and stability and contribute to ion suppression in LC-MS analysis. 105Another problem is the potential adsorption and non-specific binding of lipids to the microsampling card, given that they contain cellulose with many hydroxyl groups that can interact with the hydrophilic head of the lipid molecules. 106,107ternatively, with advances in commercially available microsampling devices, Table 1, common barriers such as volumetric inaccuracies and the haematocrit effect in DBS microsampling can be overcome in lipidomics as well.Thus, several research studies evaluate the possibility of conducting reliable -both targeted and untargeted lipidomic -studies using hemaPEN ® , 34 Tasso-M20™ 35 or qDBS Capitainer ® . 14ese devices, in addition to previous studies using VAMS, 100,108 allow the accurate collection of blood samples for comparison between traditional and advanced techniques 104 (entry 5, Table 8) as well as comparison between microsampling and venous blood collection 8,14 (entries 1 and 4, Table 8).It demonstrates the great interest of these devices to collect samples very easily on the field by the athletes themselves by using low invasive techniques and requiring less organisation.

BµS lipid extraction methods, applied approaches and instrumentation
Sample collection and preparation is probably the most critical step to ensure reliable lipidomic data collection, and only a few studies have examined the influence of lipids storage stability, recovery and reproducibility. 8,41To date, the evidence collected in Table 8 shows that there is no standardised method for lipid extraction from the different microsampling techniques.The extraction method depends on the type of lipid species being studied.For example, studies until 2021 agree that MeOH is the best solvent to extract phospholipids and fatty acids from DBSs. 36Traditionally and still today, solvent lipid extraction protocols use biphasic extraction method where lipids are commonly extracted using mixtures of water and organic solvents (mostly MTBE, CH 3 Cl and MeOH). 8,34,41,103During the last few years, attempts have been made for reducing extract complexity using single-step extraction protocols; either with two solvent combination: methanol/isopropanol or butanol mixtures -or three solvent combination such as MeOH/MTBE/CH 3 Cl mixtures. 14,35Although these last extraction methods has been evaluated and found to be best -for isolating lipids from plasma giving benefits from high coverage and simplified operation 109 -more studies are needed to determine the best solvent system so that the extraction efficiency/recovery is maximised.9).
From the seven lipidomic studies gathered in this review, there is a marked preference for targeted lipidomics analysis since it is clearly the most used approach (Table 9).The targeted approach aims to unravel specific pathways or profile-specific markers, identifying and quantifying specific lipids.From the reviewed studies, RPLC-MS with a triple quadrupole (QqQ) analyser is the most common method, and only one study 8 used FIA with a QTRAP analyser.Since we notice a current challenge in addressing the untargeted lipidomics approach, we will focus on this point.

Untargeted lipidomics
Only three lipidomic BµS studies have been found in the last year to demonstrate lipidomic changes during physical effort 35 or at different paediatric ages. 103and to evaluate the lipid recovery and sample storage stability in plasma separation cards (PSCs) versus DBSs. 104ndali et al. 35 (entry 6, Tables 8 and 9) have applied RP-HPLC coupled to MS/MS with an orbitrap to demonstrate the utility of advanced BµS, in this case, Tasso-M20™ Device to characterise metabolomic and lipidomic changes during exercise.For data analysis, an in-house standard compound library was used, and lipidomic data were analysed using LipidSearch 4.0 (Thermo Scientific), identifying a total of 118 lipids. in this study, the hierarchical clustering of the top 50 significant lipids demonstrates a clear sex-independent pattern of lipid depletion. 35 the other hand, Ferreira et al. 103 (entry 7, Tables 8 and 9) assessed the variability of the lipid profile of DBSs at paediatric ages and identified age-related variations using RP-HPLC-MS/MS.The data were identified using MS-DIAL and integrated using MZmine PSCs provide a better alternative for quantitative blood lipidomics analyses compared with DBSs. 104eat efforts have been made in this review to extract and unify the information from the articles found for lipidomic and microsampling.In this direction, the Lipidomics Standards Initiative (LSI) aims to standardise the reporting of lipidomics data and analyses and the terminology used.

SUMMARY AND OUTLOOK
Interest in BµS continues increasing as it is a convenient option for Despite the advantages of volumetric new technologies and the limitations of DBSs, such as the haematocrit effect, DBS was shown to be the preferred BµS in the studies, Mitra ® (VAMS) as the second preferred, and just a few studies evaluated the potential of other quantitative devices in comparison with DBSs, plasma or whole blood. 14,34,35,44further and deeper investigation of the developed technologies is required to promote their acceptance from research-related and non-diagnosis activities to their implementation as a clinical or patientcentred tool.Furthermore, several new devices have entered the market and will be tested for metabolomics and lipidomics applications.The collected results revealed, as previous reviews stated, 98 that more efforts are needed to standardise and harmonise results across LC-MS and to avoid significant errors in analysis and reporting.To contribute to this goal, we encourage related scientists to (i) be more specific about the description of the analytical workflow conditions, as many experimental methods focused more on the clinical and biological characteristics (ii) implement into the analysis the use of QA (quality assurance) and QC (quality control) to register changes in the analysis over time, caused by retention-time shift, degradation of the extracts or column and ion source contamination 110 and (iii) consider the integration of ISs and standard reference materials to support the accuracy and reliability of the results and (iv) continue to further stages targeting longitudinal studies with larger cohorts.
The information obtained from the reviewed lipidomic studies show its implications in both basic biology and clinical medicine, as well as informing therapeutic strategies and improving health outcomes.
The collected results have revealed, as previous reviews have already stated, 98 that more efforts are needed to standardise and harmonise results across LC-MS lipidomic platforms and avoid significant errors in analysis and reporting.Not only the increased implementation of ISs but also of standard reference materials in analytical workflows will aid in this direction.Also, actual literature of microsampling reveals that there is a need for integrate into the analysis the use of QA (quality assurance) and QC (quality control) to register changes in the instrument sensitivity over time, caused by retention-time shift, degradation of the extracts or column and ion source contamination by non-volatiles. 110Great efforts have been made in this review to extract and unify the information from the articles found for lipidomic and microsampling.In this direction, the LSI (https://lipidomicstandards.org/) aims to standardise the reporting of lipidomics data and analyses, and the terminology used.
In conclusion, different BµS have shown great promise for application in targeted and non-targeted metabolomics and lipidomics analysis.A recurring topic is the limited sample material that will be obtained by BµS.Certain analysis still requires a sufficient amount for sample preparation and pre-concentration to achieve the required limits-of-detection.However, LC-MS/MS system sensitivity is increasing as well, and developments such as the switch from analytical flow to microflow also lead to higher sensitivity.Additionally, derivatisation procedures can also help to increase the sensitivity.Analytical scientists are asked to develop new and robust analysis methods.Likewise, automation of sample preparation will become an important issue.
While automated sample preparation of liquid samples such as plasma and serum is well advanced and even used in targeted and non-targeted metabolomics studies, this needs to be improved for BµS.Different automated systems have been developed, for example, an automated DBS sampler. 111VAMS can be used with 96-well-adapter for easier handling.
a comprehensive comparison of various microsampling devices was conducted, including DBS cards (conventional DBS card (PerkinElmer-226 filter paper) and PerkinElmer-226 Bioanalysis RUO cards), volumetric DBSs (HemaXis™ DB10 collection device), VAMS technology (Neoteryx Mitra® device) and quantitative DBSs (qDBS Capitainer® B Collection device) evaluate the effectiveness of steroid profiling in DBSs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a second-tier test for congenital adrenal hyperplasia (CAH) NBS in China.The study analysed DBS samples from newborns with elevated 17-hydroxyprogesterone (17-OHP) levels detected by initial immunoassay-based screening.The steroid profiling by LC-MS/MS was further performed to measure the concentrations of 17-OHP, 21-deoxycortisol, progesterone, androstenedione and testosterone.Among 377 newborns with elevated 17-OHP, steroid profiling confirmed CAH in 171 cases (45.4%).The most common form of CAH was 21-hydroxylase deficiency (21-OHD), accounting for 154 cases (90,1%).The positive predictive value of steroid profiling for CAH was 26.0%, significantly higher than that of immunoassaybased screening alone (15.3%) and the number of false-positive results reduced by 89.1%, except in premature newborns where it is not ensured.This approach has the potential to improve the accuracy and efficiency of CAH NBS, avoiding extra costs, follow-up tests and unnecessary family anxiety associated with false positive results.The LC-MS/MS method developed is able to discriminate between the two most frequent forms of CAH, 21-OHD and 11-OHD, which helps to reduce the time of the diagnosis.The validation of the LC/MS-MS method for the quantification of steroid hormones in DBS samples

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software, allowing the identification of 156 different lipid species that belong to 11 different classes.They have found that the lipidomic signature of the three age groups (0-10 days, 2-18 months and 3-13 years) is significantly different, and the study on this profile variability in children may help to improve the knowledge of the evolution of lipid metabolism in childhood, concluding that DBSs and lipidomics can be further explored.103Finally, Bishop and Fiehn 104 (entry 5, Tables8 and 9) performed untargeted lipidomics to evaluate the lipid recovery and sample storage stability in PSCs; they also validated it for untargeted lipidomic profiling and made a quantitative comparison of PSCs versus DBSs, specifically (Noviplex™ DUO Plasma Prep Card and Whatman™ 903 Filter Paper Card).Although details for the sample extraction protocol are missing, they mention the use of RP-HPLC coupled to MS/MS using a hybrid Q-Orbitrap.They used the open-source software MS-DIAL for deconvolution, peak picking, alignment and compound identification.Also, annotated compounds were done by matching retention times, accurate precursor masses, and MS/MS fragmentation patterns against the LipidBlast library, last MS-FLO software was used to identify ion adducts, duplicate peaks and isotopic features.They identified 489 unique lipids, with 237 matching the criteria; in ESI positive ion mode, 357 lipid species and 67 labelled IS, and for ESI negative ion mode, 306 known compounds and 54 labelled IS.Overall, they found analysis in vulnerable groups and for recurrent or remote sampling; examples of this are NBS and sports monitoring and many others as demonstrated in this article.The reviewed metabolomic and lipidomic studies in 2022 and 2023 show that some of them are focused on the optimisation of the analytical workflow, verifying parameters such as recovery, reproducibility and storage stability, while other studies employed BµS for disease biomarkers identification and profiling, biological pathways understanding, developing screening methods and monitoring physical effort.In the context of target metabolites, specific applications are related to TDM, anti-doping, forensic studies and PFAS exposure.
Non-quantitative device.It is a cellulose cotton-based filter paper card, where four blood drops are collected and spread non-homogenously via capillary action.Non-quantitative device.It is a cellulose cotton-based filter paper card, where blood drops are spread non-homogenously via capillary action.Spots are preperforated in PerkinElmer-226 Bioanalysis RUO cards.https://resources.perkinelmer.com/lab-solutions/resources/docs/ifu-gr226-226-sample-collection-Non-quantitativedevice.Two or three drops of blood are applied in the middle of the device.The blood will disperse uniformly across the height blades, reducing haematocrit effect between the height samples.https://www.spotonsciences.com/hemaspot-hf/)Non-quantitative device.Five drops of blood are applied on the membrane.After one minute, the blood spread on the membrane.A desiccant is placed below the membrane, allowing a quick-drying.Quantitative device.For the 10 µL device, a drop of blood is applied to each sample well and the channels measure the right volume.For the 50 µL device, multiple drops of blood are added to the capillary until blood reaches the stop line.When the channels are filled, the blood is transferred onto the pre-punched Ahlstrom filter paper discs via capillary action.https://capitainer.com/technology/Quantitative device.Blood is collected through four EDTA-coated capillaries.When they are full, the device is reversed and clicked into its plastic base by the user.The blood is then transferred onto pre-punched Whatman or Perkin Elmer discs paper via gravity action.https://www.neoteryx.com/hemapen-dried-blood-spot-collection)Quantitative device.A drop of blood is applied to the inlet of the volumetric capillary channel.When the four channels are full, the device is closed and the blood is transferred to the Whatman or Perkin Elmer paper via capillary action.https://hemaxis.com/products/hemaxis-db10/)Quantitative device.The device is placed onto the skin of the upper arm.When the buttons are pressed, a gentle vacuum and a lancet draws the blood to two paper strips sitting in a cartridge.
TA B L E 7 8,41,104ollection and preparation (2022-2023) -lipidomics.Analytical methodology and instrumental analysis (2022-2023) -lipidomics.During sample preparation it is also common to include a drying period either by evaporation or under N 2 action, followed by a re-dissolution.Only a few recent studies investigate8,41,104the appropriate and reproducible recovery of every class of lipids, the pH, temperature, requirement of solvation energy (shaking vs. sonication) and duration of the extraction process from DBSs, and this makes appro- Abbreviations: AA, ammonium acetate; ACN, acetonitrile; AF, ammonium formate; DCM, dichloromethane; DTT, DL-dithiothreitol; EtOH, ethanol; FA, formic acid; H 2 O, water; IPA, isopropanol; IS, internal standard; MeOH, methanol; MTBE, methyl-tert-butyl ether; OX:, oxalic acid; N 2 , nitrogen; N/A, not apply; N/S, not specified.LPC, lysophosphatidylcholine; PSC, Telimmune Plasma Separation Cards.TA B L E 9 heated electrospray ionisation; N/S, not specified; PSC, Telimmune Plasma Separation Cards.a A total of 156 relevant molecular lipids, from 11 lipid subclasses, reported and identified using MS-DIAL 4 software tool based on MS/MS spectra.b A total of 118 relevant molecular lipids reported (N/S) and identified using LipidSearch 4.0 (Thermo Scientific) software tool based on accurate intact mass, isotopic pattern and fragmentation pattern.